Two-Photon fluorescence Light Microscopy

Two-Photon fluorescence Light Microscopy

Mohammad Reza SharifimehrLaser and Plasma Research Institute-SBU

April 23,2013.

Two-Photon fluorescence Light Microscopy LAPRI-SBU M.R.Sharifimehr

An Introduction to:

1/36

In The Name of GOD

Nanophotonics Course ResearchDr S.M.Hamidi

Outline

Two-Photon fluorescence Light Microscopy LAPRI-SBU M.R.Sharifimehr 2/36

An Introduction to Microscopy Classes

Nature of Two-Photon Absorption (TPA)

Difference between TPA and SHG

Comparison of Two-Photon Absorption and One-Photon Absorption

The order of required intensity for Two-Photon Excitation (TPE)

Design of a Two-Photon Microscope

Conventional Confocal and Two-Photon light microscopy Advantages and Disadvantages of TPA

Recent TPA applications

A brief history of TPA

An Example of Microscopy


Scanning electron microscope (SEM) image of pollen

Microscopy classes


Microscopy techniques has developed since 1590!

18th century microscopes

Microscopy classes


Microscopes can be separated into several different classes. One grouping is based on what interacts with the sample to generate the image

Microscopy

Optical (Photon) Microscopy(EM wave interaction with sample)

Electron Microscopy(Electron wave interaction with sample)

Scanning probe Microscopy

(force, voltage, current, … measurement, using a cantilever)

Other types

Common techniques

Microscopy classes


Microscopy





Other types

Common techniques

Dark field microscopy

Bright field microscopy

Phase contrast microscopy

Dispersion staining microscopy

Oblique illumination microscopy

Polarized light microscopy

White Light Microscopy

Fluorescence Microscopy

Confocal microscopy

Two-photon excitation microscopy

Light sheet fluorescence microscopy

Microscopy classes


Microscopy





Other types

Common techniques

Transmission Electron Microscope (TEM)

Scanning Electron Microscope (SEM)

Dark Field Microscopy

Microscopy classes


Microscopy





Other types

Common techniques

Atomic Force Microscopy (AFM)

Electrostatic Force Microscopy (EFM)

Scanning Tunneling Microscopy (STM)

Scanning Thermal Microscopy (SThM)

Scanning Capacitance Microscopy (SCM)

Scanning Near-Field Optical Microscopy(SNOM)

Near-Field Scanning Optical Microscopy(NSOM )

Microscopy classes


Microscopy





Other types

Common techniques

X-ray microscopy

Scanning acoustic microscopes

Ultrasonic Force Microscopy (UFM)

digital holographic microscopy (DHM)

Microscopy classes


Microscopy





Other types

Common techniques

Microscope Image Processing

Digital Microscopy (CCD+Microscope)

Multifocal Plane Microscopy (Multiplane Microscopy)

An Example of Fluorescence Microscopy


The microtubules are red DNA is stained blue A protein called INCENP is green

An Example of Fluorescence Microscopy

Two-Photon fluorescence Light Microscopy LAPRI-SBU M.R.Sharifimehr 12/36fluorescent imaging of the human cancer cell

++

Fluorescence Microscopy

Two-Photon fluorescence Light Microscopy LAPRI-SBU M.R.Sharifimehr 13/36Schematic of a fluorescence microscope

A brief history of Two-photon fluorescence light microscopy:


1929Maria Goppert -Mayer- Theoretical basis of two-photon excitation was established

1963Kaiser and Garret - two-photon excitation was verified experimentally

1990Denk et al. - The invention of two-photon fluorescence light microscopy

Theory --> Experiment --> Application

A brief history of Two-photon fluorescence light microscopy:


Milestones relevant to the development of two-photon microscopy

Two-Photon Absorption and Fluorescence Definition


The familiar one-photon fluorescence process involves exciting a fluorophore from the electronic ground state to an excited state by a single photon. This process typically requires photons in the ultraviolet or blue/green spectral range. However, the same excitation process can be generated by the simultaneous absorption of two less energetic photons (typically in the infrared spectral range) under sufficiently intense laser illumination. This nonlinear process can occur if the sum of the energies of the two photons is greater than the energy gap between the molecule’s ground and excited states. Under sufficiently intense excitation, three-photon and higher-photon excitation is also possible and deep UV microscopy based on these processes has been developed.

Two Photon Absorption (TPA) Process


Jablonski diagram of one-photon (a) and two-photon (b) excitation, which occurs as fluorophores are excited from the ground state to the first electronic states. One-photon excitation occurs through the absorption of a single photon. Two-photon excitation occurs through the absorption of two lower-energy photons via short-lived intermediate states. After either excitation process, the fluorophore relaxes to the lowest energy level of the first excited electronic states via vibrational processes. The subsequent fluorescence emission process for both relaxation modes is the same.

Difference between TPA and SHG


Two-Photon Excitation (TPE) is different from SHG, because in SHG the optical output is fixed at 2ω and is a very sharp line at 2ω, related to the line width only of the fundamental at frequency ω. In the TPE, ω3 is a higher frequency compared to either ω1 or ω2, but it is not 2ω1, 2ω2, or ω1 + ω2. In most cases, ω3 < (ω1+ ω2).

TPA Equations


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Two (Multi)-Photon Absorption versus One-Photon Absorption


A fluorophore that is one-photon active at wavelength λ can often be excited by two photons of twice the wavelength (2λ).

One-photon and two-photon excitation are fundamentally different quantum-mechanical processes and have very different selection rules.

A fluorophore’s two-photon excitation spectrum scaled to half the wavelength is typically not equivalent to its one-photon excitation spectrum. Further, fluorophores designed for one-photon excitation are not necessarily optimized for good two-photon absorption characteristics (such as σ).

A fluorophore’s emission spectrum, is independent of the excitation mechanism, since the molecule relaxes to the same excited state through vibrational mechanisms before emission.

Two (Multi)-Photon Absorption versus One-Photon Absorption


Comparison of one-photon (broken lines) and three-photon (solid lines) fluorescence excitation

Other differences between one-photon and two-photon excitation


For a spatially uniform specimen, fluorescence signals are generated equally from each z-section above and below the focal plane for one-photon excitation. In contrast, over 80% of the total fluorescence signal can be confined to a region 1µm thick about the focal point using two-photon excitation. This property is a base for depth discrimination.

A demonstration of the localization of two-photon excitation volume

Two Photon Absorption (TPA) Process


Question:

What’s the order of required intensity for two-photon excitation?

Answer:

A high-radiance light source on the order of is required for efficient two-photon excitation .

21210 /1010 cmw

High repetition rate (100 MHz), ultrafast (femtosecond or picosecond pulse widths) lasers, such as titanium–sapphire and Nd:YLF lasers, are the most widely used light sources.

Confocal versus Two-Photon Light Microscopy


Confocal microscopy is a technique very similar to two-photon microscopy.

Conventional Light microscope --> Confocal Microscopy (1960s) --> Two-Photon Microscopy (1990s) --> 3D Imaging

Two-photon excitation wavelengths are typically about twice the one-photon excitation wavelengths. This wide separation between excitation and emission spectrum ensures that the excitation light and the Raman scattering can be rejected while filtering out a minimum of fluorescence photons.

Near-infrared radiation used in two-photon excitation has orders of magnitude less absorption in biological specimens than UV or blue-green light. The attenuation of excitation light from scattering is also reduced, as the scattering cross-section decreases with increasing wavelength.

The resolution of a microscope system scales inversely with the wavelength of light used. For a given fluorophore, two-photon excitation requires the use of excitation at twice the one-photon wavelength, resulting in approximately half the resolution.

Confocal versus Two-Photon Light Microscopy


The reduction in the photodamage volume results in a dramatic increase in viability of biological specimens.

Confocal microscopy obtains 3D resolution by limiting the observation volume, whereas two-photon microscopy limits the excitation volume.

Compared with confocal microscopy operating in the UV or blue-green excitation wavelengths, two-photon microscopy minimizes photobleaching and photodamage. The reduction in the photodamage volume results in a dramatic increase in viability of biological specimens.

Typical two-photon microscopy design


A schematic drawing of typical components in a two-photon microscope. This system typically consists of a high-peak-power pulsed laser, a high-throughput scanning microscope and high-sensitivity detection circuitry.

Galvo Mirrors

?Barrier filter

TPA Imaging Disadvantages


A critical component in a two-photon microscope is its light source and very kind of light sources can’t be use in this method.

A High-sensitivity detection electronics, such as single-photon counting circuitry, is needed to ensure maximal detection efficiency.

Photodamage maybe caused by dielectric breakdown, resulting from the intense electromagnetic field of the femtosecond laser pulses.

My Experiment


Concept --> Experiment --> Application

My Experiment

Typical two-photon microscopy-Image Construction


3D Images are constructed by raster scanning the fluorescent volume in three dimensions using a galvanometer driven x–y scanner and a piezo-objective z-driver.

A two-photon laser-scanning fluorescence microscopy 3D image of sulphorhodamine-stained human epidermis in vitro.

A voxelgram generated from a two-photon image stack of a GFP-labeled cortical pyramidal neuron.

TPA microscopes


LSM 710 NLO & LSM 780 NLO

http://microscopy.zeiss.com

Carl Zeiss (over 160 years): Laser scanning microscope for multiphoton imaging

TPA microscopes


Movie 1

scanning microscope for multiphoton imaging

Recent TPA Application


- High-speed 3D printing

- Two-Photon Microscopy for 4D Imaging of Living Neurons

- 3D Photonic crystals Fabrication using Two-Photon lithography

- Single Molecule Detection



Two-Photon Microscopy for 4D Imaging of Living Neurons

Movies 2



3D Photonic crystals Fabrication using Two-Photon lithography

Tightly focused in a photosensitive material, femtosecond laser pulses initiate two-photon polymerization and produce structures with a resolution down to 200 nm.

SEM image of a photonic crystal fabricated by two-photon lithography. From Cumpston et al. (1999)

SEM image of structures produced by two-photon polymerization of Deso-Bond 956-105. A resolution of ~200 nm was achieved.



High-speed 3D printing of tiny objects:

A race car model no larger than a grain of sand, created using the new high-speed two-photon lithography process

A model of St. Stephen's Cathedral, Vienna, created using the new high-speed two-photon lithography process

A model of London's Tower Bridge, created using the new high-speed two-photon lithography process



Single Molecule Detection

Single emitting molecules are currently providing a new window into nanoscale systems ranging from biology to materials science. The amount of information that can be extracted from each single molecule depends upon the specific photophysical properties of the fluorophore and how these properties are affected by the nearby environment.

Single Molecules and Nanotechnology- R. Rigler-2008, Springer:

Two-Photon Fluorescence scanning confocal image of single DCDHF-6 molecules in a PMMA film.



Single-molecule spectroscopy has two requirements:

1-There is only one molecule present in the volume probed by the light source. This condition is met by using appropriate dilution together with microscopic techniques to probe a small volume.

2-The signal-to-noise (SNR) ratio for the single-molecule signal is sufficiently greater than unity for a reasonable averaging time to provide adequate sensitivity. For this purpose, large absorption cross sections, high photostability, operation below saturation of absorption, and (in the case of fluorescence detection) a high fluorescence quantum yield are needed.

Fluorescence NSOM images of single molecules.

References


Movies 3

We need a powerful software for Image processing

References


[2] Two-Photon Fluorescence Light Microscopy-(2002),Peter TC So- ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group

[4] Multiphoton Fluorescence Light Microscopy -15th June 2012, Konstantinos Palikaras-John Wiley & Sons, Ltd

[5] Nanophotonics- Paras N. Prasad, 2004, John Wiley & Sons- Chapters 3, 9, 11

[6] Properties of single organic molecules on crystal surfaces, Peter Grütter, 2006-Imperial College Press

[7] Nonlinear and Two-Photon-Induced Fluorescence, JOSEPH R. LAKOWICZ, 2002 Kluwer Academic Publishers

[8] Photophysics of Molecular Materials, Guglielmo Lanzani, 2006 WILEY-VCH Verlag GmbH

[9] Photonics, Ralf Menzel, 2006 Springer

[10] Nonlinear Optics, second edition, Robert W..Boyd, 2003

[1] Topics in Fluorescence Spectroscopy- Volume 5- Nonlinear and Two-Photon-Induced Fluorescence-Joseph R. Lakowicz-kluwer academic publishers

[3] Single Molecules and Nanotechnology- R. Rigler-2008, Springer

ThanksTwo-Photon fluorescence Light Microscopy LAPRI-SBU M.R.Sharifimehr 36/36

There is a big difference between knowing and understanding. For example, you can “know” the sky is blue but “understanding” why it’s blue is so much more important.

Two-Photon fluorescence Light Microscopy

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